WO1996041009A1 - Restes permettant de lier des troisiemes brins a des complexes doubles nucleotidiques complementaires de n'importe quelle sequence paire de base - Google Patents

Restes permettant de lier des troisiemes brins a des complexes doubles nucleotidiques complementaires de n'importe quelle sequence paire de base Download PDF

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Publication number
WO1996041009A1
WO1996041009A1 PCT/US1996/009428 US9609428W WO9641009A1 WO 1996041009 A1 WO1996041009 A1 WO 1996041009A1 US 9609428 W US9609428 W US 9609428W WO 9641009 A1 WO9641009 A1 WO 9641009A1
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residue
backbone
oligonucleotide
bases
strand
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PCT/US1996/009428
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English (en)
Inventor
Jacques R. Fresco
Bin Lin
Lynn C. Klotz
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Princeton University
Oncorpharm, Inc.
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Application filed by Princeton University, Oncorpharm, Inc. filed Critical Princeton University
Priority to AU62601/96A priority Critical patent/AU6260196A/en
Priority to EP96921358A priority patent/EP0871772A4/fr
Publication of WO1996041009A1 publication Critical patent/WO1996041009A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays

Definitions

  • the invention relates to compositions of matter capable of serving as residues for specific binding of third strands to double-stranded complementary nucleic acids of any base-pair sequence.
  • Canonical base any one of the standard nucleic acid bases, adenine-A, guanine-G, cytosine-C, thymine-T, uracil- U. - • Canonical base pair - the complementary or Watson- '
  • Inverted base pair - a target base pair with its pyrimidine base located in a purine-rich strand of a target duplex sequence, i.e., U/TA, CG.
  • An inverted base pair therefore interrupts the continuity of a homopurine strand sequence.
  • Triplex motif - triplex stereochemistry as determined by the predominant bases of the third strand binding to a target Watson-Crick duplex with purine-rich, pyrimidine- rich strands and by the orientation of the third strand relative to that of the purine-rich strand of the target. Note that the mode of third strand base or residue H- bonding to the target base pair is characteristic of each motif.
  • W-residue - a synthetic third strand residue designed to bind with specificity to a particular direct target base pair or inverted target base pair.
  • Oligonucleotides (third strands) can bind to double- stranded nucleic acids to form triple-stranded helices (triplexes) in a sequence specific manner (Beal and Dervan, Science 251: 1360 (1991); Beal and Dervan, Nucleic Acids Res. , 20:2773 (1992); Broitman and Fresco, Proc. Natl . Acad. Sci . USA, 84:5120 (1987); Fossella, et al . , Nucleic . Acid ⁇ Res. 21:4511 (1993); Letai et al., Biochemistry 27:9108 (1988); Sun, et al . , Proc. Natl . Acad. Sci . USA 86:9198 (1989)).
  • the third strand binding code (a complementarity principle) dictates the sequence specificity for binding third strands in the major groove of double-stranded nucleic acids to form a triple-stranded helix or triplex'.
  • the code provides the specificity of third-strand binding for design of gene-based therapeutics that bind specifically to target nucleic acid sequences with little or no non-specific binding to non-target sequences.
  • Third-strand binding differs from the familiar Watson- Crick complementarity principle (A:T/U and G:C) for the double-stranded helix in two major respects: (1) the third-strand binding code is degenerate, and (2) third strands bind only to double-strands which contain a sequence of adjacent (or run of) purine bases (A or G) in one of the strands, which here will be called the center or core strand.
  • the third-strand binding code is illustrated in the Table 1 below. Table 1
  • a "+" means the bases are complementary or correspondent, and a "-" means they are not complementary or not correspondent.
  • a serious practical limitation for stable third-stand binding dictated by the code in Table 1 is the necessity for runs of purines in the center target strand of typically 10 or more bases interrupted by only one or two pyrimidines (hereafter called "purine-rich" sequences or” targets) . While runs of sufficient length are present in many of the genes and the non-gene DNA (or RNA) of eukaryotes and prokaryotes and their viruses, they are not frequent enough for widespread diagnostic and therapeutic uses. It is therefore desirable to be able to target a duplex nucleic acid segment with a mixed purine and pyrimidine composition in the center strand.
  • motifs which further define third-strand binding to purine-rich targets, still in conformity with the third-strand binding code.
  • the motifs define the base-compositional features of the third strand and whether the third-strand binds parallel or antiparallel to the purine-rich target strand (polarity) . Motifs thereby define the hydrogen-bonding (H-bonding) schemes of the third-strand bases to the target base pairs.- In consequence, the motifs also determine target specificity and nearest neighbor effects on binding.
  • There are five motifs that describe third-strand binding (Sun and Helene, Current Opinion in Structural Biology. 3:345 (1993)) . Table 2 summarizes the five motifs, which constitute a subset of constraints to the binding code.
  • the motifs provide further instructions for defining the sequences of different third-strands that can alternatively bind with specificity to the same target.
  • the Table also gives examples of selected analog bases which may be substituted for the standard or canonical A, G, T/U and C third-strand bases.
  • the colon indicates third-strand binding of the base to the left of the colon, to at least the center purine base on the immediate right of the colon.
  • the + superscript indicates that the base is in the protonated form when it binds (the energy of binding provides the energy for protonation) .
  • 2,6 DAP stands for 2,6 diaminopurine.
  • Parallel or antiparallel refers to the relation of third-strand polarity in the triplex relative to the purine-rich target strand.
  • t .re are other considerations that can affect the stability of the resulting triplex.
  • Third strands composed of A and G bases for example, have the potential.for several kinds of self structure. Very G-rich third strands tend to form either hairpin or linear helices stabilized by G-tetrads (Fresco and Massoulie, J. Am. Chem. Soc , 85:1352 (1963); Zimmerman, et al . , J. Mol . Biol . , 92:181 (1975); Gem, et al . Biochemistry, 34:2042 (1994)).
  • U.S. Patent Nos. 5,034,506, 5,166,315 and 5,405,938 are directed to various polymers said to be effective to bind to Watson-Crick base pairs.
  • those patents do not precisely model the stereochemistry of the canonical base triplets; nor do they precisely model the stereochemistry of their designed residues ⁇ Instead, they are directed to flexible non-native backbones that, upon triplex formation, are possibly capable of assuming locations acceptable for triplex formation.
  • backbones of greater flexibility than native sugar-phosphate backbones may suffer unacceptable negative entropy changes (positive free energy changes) when they are "frozen" into the helical configuration demanded by triplex stereochemistry. This energetically unfavorable change may prevent stable triplex formation.
  • those patents show residues only for the pyrimidine/parallel motif, not for the other four known triplex motifs.
  • WO 94/11534 is directed to third-strand residues which have been designed to bind to modified inverted duplex base pairs.
  • the designed residues bind only to the core or center strand duplex base; and to do so, the center strand base must be modified to possess two hydrogen bonding sites.
  • duplex base modification to accomplish third-strand binding makes the invention of little use; in particular, it can have no use in therapeutics, since any disease target from a living organism would not possess the required modified bases that are the targets for such triplex formation.
  • the present invention relates to rules and guidelines for designing heterocycles and other structures, and to compositions of matter ("residues") designed by those processes that when incorporated into third strands (with natural or synthetic backbones) make those strands capable of specific binding to complementary double-stranded nucleic acids of any base-pair sequence; that is, without the target requiring a purine-rich strand.
  • a major object of the present invention is to provide synthetic nucleic acid monomers ("residues”), that when incorporated into an oligonucleotide ("third strand"), or analog oligomer, i . e . , a third strand with a synthetic backbone, enables the third strand to form a triple-stranded nucleic acid ("triplex") when hybridized to a double-stranded nucleic acid (“duplex”), wherein the "target region" to which the third strand binds is of substantially any base sequence; that is, it need not include a run of a large number of adjacent purines on one strand. In other words, the residues that are provided will be capable of strong and specific binding to inverted base pairs.
  • the present invention provides a partially synthetic oligonucleotide containing a backbone having a polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide capable of binding in a parallel orientation relative to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising pyrimidine bases and/or base analogs ' thereof, and said residue(s) being substantially planar, such that when the oligonucleotide binds to the target sequence, base triplets are formed among each oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone which is linked to the corresponding residue of the oligonucleotide is from about 7.0 A to about 8.6 A;
  • the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
  • the present invention provides a partially synthetic oligonucleotide containing a backbone having a polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide capable of binding in a parallel orientation relative to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue(s) being substantially planar, such that when the oligonucleotide binds to the target sequence, base triplets are formed among each oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone which is linked to the corresponding residue of the oligonucleotide is from about 6.7 A to about 8.6 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone which is bound to the residue is from about 81° to about 125°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone, and the bond vector between the residue and the oligonucleotide backbone is from about -100° to about +60°; and d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
  • the present invention provides a partially synthetic oligonucleotide containing a backbone having a polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide capable of binding in an antiparallel orientation relative to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue( ⁇ ) being substantially planar, such that when the oligonucleotide binds to the target sequence, base triplets are formed among each oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters: '
  • the radius of the imaginary circle connecting the Cl*' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone which is linked to the corresponding residue of the oligonucleotide is from about 6.5 A to about 8.6 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone which is bound to the residue is from about 86° to about 128°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone, and the bond vector between the residue and the oligonucleotide backbone is from about -45° to about +120°; and d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
  • the present invention provides a partially synthetic oligonucleotide containing a backbone having a polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide capable of binding in a parallel orientation relative to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil and/or base analogs thereof, or 2, 6-diaminopurine to bind to AT/U base pairs, and said residue(s) being substantially planar, such that when the oligonucleotide binds to the target sequence, base triplets are formed among each oligonucleotide base or residue and the corresponding bases of
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone which is linked to the corresponding residue of the oligonucleotide is from about 7.0 A to about 8.6 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone which is bound to the residue is from about 62° to about 107°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone, and the bond vector between the residue and the oligonucleotide backbone is from about -90° to about +90°; and
  • the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
  • the present invention provides a partially synthetic oligonucleotide containing a backbone having a polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide capable of binding in an antiparallel orientation relative to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil and/or base analogs thereof or 2, 6-diaminopurine to bind to AT/U base pairs, and said residue(s) being substantially planar, such that when the oligonucleotide binds to the target sequence, base triplets are formed among each oligonucleotide base or residue and the corresponding bases
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone which is linked to the corresponding residue of the oligonucleotide is from about 5.9 A to about 8.2 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone which is bound to the residue is from about 90° to about 110°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone, and the bond vector between the residue and the oligonucleotide backbone is from about -30° to about +120°; and d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
  • the present invention provides a substantially synthetic oligonucleotide analog containing a backbone having no polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide analog being effective to bind in a sequence- specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide analog capable of binding to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising pyrimidine bases and/or base analogs thereof, and said residue(s) being substantially planar, such that when the oligonucleotide analog binds to the target sequence, base triplets are formed among each analog oligonucleotide base or. residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide analog backbone which is linked to the corresponding residue of the oligonucleotide analog is from about 7.0 A to about 8.6 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide analog backbone which is bound to the residue is from about 53° to about 82°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide analog backbone, and the bond vector between the residue and the oligonucleotide analog backbone is from about. -90° to about +90°; and d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the du
  • the present invention provides a substantially synthetic oligonucleotide analog containing a backbone having no polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide analog being effective to bind in a sequence- specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide analog capable of binding to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue(s) being substantially planar, such that when the oligonucleotide analog binds to the target sequence, base triplets are formed among each analog oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide analog backbone which is linked to the corresponding residue of the oligonucleotide analog is from about 6.7 A to about 8.6 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide analog backbone which is bound to the residue is from about 81° to about 125°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide analog backbone, and the bond vector between the residue and the oligonucleotide analog backbone is from about -100° to about +60°; and d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the du
  • the present invention provides a substantially synthetic oligonucleotide analog containing a backbone having no polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide analog being effective to bind in a sequence- specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide analog capable of binding to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue(s) being substantially planar, such that when the oligonucleotide analog binds to the target sequence, base triplets are formed among each analog oligonucleotide base or residue and the corresponding bases of the target base pair of the duplex, and each residue conforms to the following parameters:
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide analog backbone which is linked to the corresponding residue of the oligonucleotide analog is from about 6.5 A to about 8.6 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide analog backbone which is bound to the residue is from about 86° to about 128°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide analog backbone, and the bond vector between the residue and the oligonucleotide analog backbone is from about -45° to about +120°; and d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the du
  • the present invention provides a substantially synthetic oligonucleotide analog containing a backbone having no polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide analog being effective to bind in a sequence- specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide analog capable of binding to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil or base analogs thereof or 2,6,-diaminopurine to bind to AT/U base pairs, and said residue(s) being substantially planar, such that when the oligonucleotide analog binds to the target sequence, base triplets are formed among each analog oligonucleotide base or residue and the corresponding bases of the duplex,
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide analog backbone which is linked to the corresponding residue of the oligonucleotide analog is from about 7.0 A to about 8.6 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide analog backbone which is bound to the residue is from about 62° to about 107°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide analog backbone, and the bond vector between the residue and the oligonucleotide analog backbone is from about -90° to about +90°; and d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the du
  • the present invention provides a substantially synthetic oligonucleotide analog containing a backbone having no polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide analog being effective to bind in a sequence- specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide analog capable of binding to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil and/or base analogs thereof or 2, 6-diaminopurine to bind to AT/U base pairs, and said residue(s) being substantially planar, such that when the oligonucleotide analog binds to the target sequence, base triplets are formed among each analog oligonucleotide base or residue and the corresponding bases of the duplex
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide analog backbone which is- linked to the corresponding residue of the oligonucleotide analog is from about 5.9 A to about 8.2 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide analog backbone which is bound to the residue is from about 90° to about 110°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide analog backbone, and the bond vector between the residue and the oligonucleotide analog backbone is from about -30° to about +120°; and d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
  • the present invention provides a method of forming a triple-stranded nucleic acid comprising the steps of: a) providing a nucleic acid core or center strand which has a target sequence with 50% or more of purine bases; b) providing a complementary nucleic acid strand which is hydrogen bonded in a Watson-Crick manner to said target sequence on the core or center strand; c) providing a third nucleic acid oligonucleotide or backbone analog strand comprising a natural or synthetic backbone, in the latter case that is directional or nondirectional, containing nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said third strand being effective to bind in a sequence-specific manner to the target sequence, said nucleotide bases comprising pyrimidine bases and/or base analogs thereof, and said residue(s) being substantially planar; and d) contacting duplex formed from said core or center and complementary strands with said third strand, so as
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone or backbone analog which is linked to the corresponding residue of the oligonucleotide or backbone analog is from about 7.0 A to about 8.6 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone or backbone analog which is bound to the residue is from about 53° to about 82°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone or backbone analog, and the bond vector between the residue- and the oligonucleotide backbone or backbone analog is from about -90° to about +90°; and iv) the residue forms a total of at
  • the present invention provides a method of forming a triple-stranded nucleic acid comprising the steps of: a) providing a nucleic acid core or center strand which has a target sequence with 50% or more of purine bases; b) providing a complementary nucleic acid strand which is hydrogen bonded in a Watson-Crick manner to said target sequence on the core or center strand; c) providing a third nucleic acid oligonucleotide or backbone analog strand comprising a natural or synthetic backbone, in the latter case that is directional or nondirectional, containing nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said third strand being effective to bind in a sequence-specific manner to the target sequence, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue(s) being substantially planar; and d) contacting duplex formed from said core or center and complementary strands with said third strand, so as to allow
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone or backbone analog which is linked to the corresponding residue of the oligonucleotide or backbone analog is from about 6.7 A to about 8.6 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone or backbone analog which is bound to the residue is from about 81° to about 125°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone or backbone analog, and the bond vector between the residue and the oligonucleotide backbone or backbone analog is from about -100° to about +60°; and iv) the residue forms a total of at
  • the present invention provides a method of forming a triple-stranded nucleic acid comprising the steps of: a) providing a nucleic acid core or center strand which has a target sequence with 50% or more of purine bases; b) providing a complementary nucleic acid strand which is hydrogen bonded in a Watson-Crick manner to said target sequence on the core or center strand; c) providing a third nucleic acid oligonucleotide or oligonucleotide analog strand comprising a natural or synthetic backbone, in the latter case that is directional or nondirectional, containing nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said third strand being effective to bind in a sequence-specific manner to the target sequence, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue(s) being substantially planar; and d) contacting duplex formed from said core or center and complementary strands with said third
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone or backbone analog which is linked to the corresponding residue of the oligonucleotide or backbone analog is from about 6.5 A to about 8.6 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone or backbone analog which is bound to the residue is from about 86° to about 128°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone or backbone analog, and the bond vector between the residue and the oligonucleotide backbone or backbone analog is from about -45° to about +120°; and iv) the residue forms a total of at
  • the present invention provides a method of forming a triple-stranded nucleic acid comprising the steps of: a) providing a nucleic acid core or center strand wh'ich has a target sequence with 50% or more of purine bases; b) providing a complementary nucleic acid strand which is hydrogen bonded in a Watson-Crick manner to said target sequence on the core or center strand; c) providing a third nucleic acid oligonucleotide or oligonucleotide analog strand comprising a natural or synthetic backbone, in the latter case that is directional or nondirectional, containing nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said third strand being effective to bind in a sequence-specific manner to the target sequence, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil and/or base analogs thereof, or 2, 6-diaminopurine to
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone or backbone analog which is linked to the corresponding residue of the oligonucleotide or backbone analog is from about 7.0 A to about 8.6 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone or backbone analog which is bound to the residue is from about 62° to about 107°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone or backbone analog, and the bond vector between the residue and the oligonucleotide backbone or backbone analog is from about -90° to about +90°; and iv) the residue forms a total of at
  • the present invention provides a method of forming a triple-stranded nucleic acid comprising the steps of: a) providing a nucleic acid core or center strand which has a target sequence with 50% or more of purine bases,* b) providing a complementary nucleic acid strand which is hydrogen bonded in a Watson-Crick manner to said target sequence on the core or center strand; c) providing a third nucleic acid oligonucleotide or oligonucleotide analog strand comprising a natural or synthetic backbone, in the latter case that is directional or nondirectional, containing nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said third strand being effective to bind in a sequence-specific manner to the target sequence, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil and/or base analogs thereof or 2,6-diaminopurine to bind
  • the radius of the imaginary circle connecting the Cl' ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone or backbone analog, which is linked to the corresponding residue of the oligonucleotide or backbone analog is from about 5.9 A to about 8.2 A;
  • the ⁇ value, measured from the Cl' atom bound to the base in the core or center strand, to the atom of the oligonucleotide or backbone analog which is bound to the residue is from about 90° to about 110°;
  • the ⁇ value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide or backbone analog, and the bond vector between the residue and the oligonucleotide or backbone analog is from about -30° to about +120°; and iv) the residue forms a total of at least two hydrogen bonds with one or both
  • Figure 1 depicts the standard ring numbering scheme for purines and pyrimdines.
  • Figure 2 is a two-dimensional illustration, drawn to scale, of base triplet hydrogen bonding (H-bonding) , imaginary D-circle centers and radii for canonical pyrimidine/parallel motif base triplets T:AT and C + :GC.
  • FIG. 3 is a two-dimensional illustration drawn to scale of base triplet H-bonding, D-circle centers and radii for canonical purine/parallel motif base triplets A:AT and G:GC.
  • base triplet H-bonding D-circle centers and radii for canonical purine/parallel motif base triplets A:AT and G:GC.
  • Table 7 For each third-strand base, two H-bonds to the target direct base pair were placed at standard distances (Table 7) and are essentially linear or well within the 25° preferred bending limit from linearity.
  • the G:GC triplet requires an intermediary H 0 molecule both to form the triplet and to achieve preferred substantially identical glycosyl-bond position and orientation for both triplets.
  • the "x" and "o" symbols at the ends of the glycosyl bonds denote parallel and antiparallel backbone directions, respectively. Model drawing errors are estimated to be about 3% of measured distances.
  • Figure 4 is a two-dimensional illustration, drawn to scale, of base triplet H-bonding, D-circle centers and radii for canonical purine/antiparallel motif base triplets A:AT and G:GC.
  • the H-bonds were placed at standard distances (Table 7), with all but one H-bond essentially linear or well within the 25° preferred bending limit from linearity.
  • One H-bond, the N H-N involving the Ni of the third strand A base has a 25° bend, at the limit of the preferred range for bending.
  • the D-circle radii are slightly different for the A:AT and G:GC triplets, 7.2 A and 7.6 A, respectively.
  • Figure 5 is a two-dimensional illustration, drawn to scale, of base triplet H-bonding, D-circle centers and radii for canonical T and G/parallel motif base triplets T:AT and G:GC.
  • the T:AT triplet is identical to that of Figure 2
  • the G:GC triplet is identical to that of Figure 3.
  • the D-circle radii are 8.0 A and 7.8 A for T:AT and G:GC triplets, respectively.
  • the orientations of the glycosyl bonds are similar, 0° and -21° for T:AT and G:GC, respectively.
  • Figure 6 is a two-dimensional illustration, drawn to scale, of base triplet H-bonding, D-circle centers and radii for canonical T and G/antiparallel motif base triplets T:AT and G:GC.
  • the H-bonds for- T:AT were placed at standard distances (Table 7), and are essentially linear, well within the 25° preferred bending limit from linearity.
  • the G:GC triplet is identical to that of Figure 3.
  • the D-circle radius for T:AT is quite small, 6.4 A, and quite different than that for G:GC, 7.6 A.
  • the positions of the third-strand base glycosyl bonds are identical,
  • FIG. 7 is an illustration, drawn to scale, of the definitions of D-circle radius, glycosyl-bond position angle, ⁇ , and glycosyl-bond orientation angle, ⁇ .
  • the D-circle radius denoted by r D
  • r D is that of the unique circle which can be drawn through three points, which in the invention are the three Ci' deoxyribose atoms of the three glycosyl bonds.
  • the center of the circle is located at the intersection of the perpendicular bisectors of any two of the three lines connecting two of the three points. The radius is then the distance from the center to any of the three points.
  • the ⁇ value represents the angular displacement of the Ci' atom of the third-strand base from the center or core third-strand residue on the D-circle.
  • ⁇ is measured clockwise from the Ci' atom of the purine R to the Ci' atom of the third-strand Z base.
  • the ⁇ values represent the direction of the glycosyl bond of each triplet base Z.
  • ⁇ is measured clockwise as the angle between the D-circle radius and the glycosyl bond origin at the Ng or Ni atoms of the purine or pyrimidine bases, respectively, or in the case of W residues (i.e., the synthetic residues in accordance with the present invention) , of whatever atom of the heterocycle the sugar is linked to.
  • Figure 8 is an illustration, drawn to scale, of the inverted TA and CG base pairs. Contrast with the direct AT and GC base pairs in Figure 2.
  • Figure 9 is a schematic illustration, drawn to scale, summarizing the acceptable range for D-circle radius, ⁇ and ⁇ for the pyrimidine/parallel motif for designed residues
  • Figure 10 is a schematic illustration, drawn to scale, summarizing the acceptable range for D-circle radius, ⁇ and ⁇ for the purine/parallel motif for designed residues W.
  • the only molecular details shown are the glycosyl bonds, which are drawn with correct length and orientations.
  • the positions of the glycosyl bonds for the canonical base-pairs RY, are denoted by “x" and "o” as in previous figures.
  • the positions of the glycosyl bonds for the canonical third-strand bases A and G are denoted by those base letters. Note that the A and G third-strand base glycosyl bonds lie at somewhat different positions and on different circles, but have very similar orientations in this motif.
  • the area surrounded by dots substantially encompasses the acceptable area in which the Cl' end of the glycosyl bond of designed W residues can reside, derived from the acceptable ranges in Tables 4, 5 and 6.
  • Figure 12 is a schematic illustration, drawn to scale, summarizing the acceptable range for D-circle radius, ⁇ and ⁇ for the T and G/parallel motif for designed residues W.
  • the only molecular details shown are the glycosyl bonds, which are drawn with correct length and orientations.
  • the positions of the glycosyl bonds for the canonical base-pairs RY, are denoted by “x" and "o * " as in previous figures.
  • the positions of the glycosyl bonds for the canonical third-strand bases T and G are denoted by those base letters. Note that the T and G third-strand base glycosyl bonds lie at substantially different positions but on substantially the same D-circles, and with similar orientations in this motif.
  • the area surrounded by dots substantially encompasses the acceptable area in which the Cl ' end of the glycosyl bond of the designed W residues can reside, derived from the acceptable ranges in Tables 4, 5 and 6.
  • Figure 13 is a schematic illustration, drawn to scale, summarizing the acceptable range for D-circle radius, ⁇ and ⁇ for the T and G/antiparallel motif for designed residues
  • the only molecular details shown are the glycosyl bonds, which are drawn with correct length and orientations.
  • the positions of the glycosyl bonds for the canonical base-pairs RY, are denoted by “x" and "o" as in previous figures.
  • the positions of the glycosyl bonds for the canonical third-strand bases T and G are denoted by those base letters. Note that the T and G third-strand base glycosyl bonds lie at substantially the same position and with similar orientations, but on substantially different D-circles in this motif.
  • the area surrounded by dots substantially encompasses the acceptable area in which the Cl' end of the glycosyl bond of the designed W residues can reside, derived from the acceptable ranges in Tables 4, 5 and 6.
  • Figure 14 depicts two three-ring carbon-nitrogen heterocycles, one four-ring carbon-nitrogen heterocycle, and one two-ring carbon-nitrogen heterocycle as examples of suitable residues according to the present invention, drawn to scale.
  • Figure 15 depicts the designed three-ring heterocycle residue, designated Fl, that binds to the inverted CG base- pair in the pyrimidine/parallel and T and G/parallel motifs.
  • the F1:CG triplet modelled in two-dimensions and drawn to scale superimposes on the acceptable-area diagrams for the pyrimidine/parallel (shown in Figure 15(a)) and T and G/parallel (shown in Figure 15(b)) motifs.
  • the glycosyl bond is attached to N 9 of Fl, with Fl in the anti-configuration about the ribose.
  • the end of the Fl glycosyl bond is denoted by a square with an x to signify that Fl may be synthesized to have its backbone either parallel or antiparallel to the center strand (with the cytosine base) .
  • the parallel orientation applies.
  • the Fl residue was positioned by placing the third-strand H-bonds at distances indicated in Table 7 for the types of H-bond involved.
  • Figure 16 depicts the designed three-ring heterocycle residue, designated F2, that binds to the inverted TA base pair in the purine/parallel and purine/antiparallel motifs.
  • the F2:TA triplet modelled in two-dimensions and drawn to scale superimposes on the acceptable-area diagrams for the purine/parallel (shown in Figure 16(a)) and purine/antiparallel (shown in Figure 16(b)) motifs.
  • the glycosyl bond is attached to the N 7 position-of F2, with F2 in the anti-configuration about the ribose.
  • the end of the F2 glycosyl bond is denoted by a square with an x to signify that F2 may be synthesized to have its backbone either parallel or antiparallel to the center strand (with the thymine base) .
  • both the parallel and antiparallel orientations apply.
  • the F2 residue was positioned by placing the third-strand H-bonds at distances indicated in Table 7 for the types of H-bond involved.
  • Figure 17 depicts the designed four-ring heterocycle residue, designated F3, that binds to the inverted TA base pair in the pyrimidine/parallel and T and G/parallel motifs.
  • the F3:TA triplet modelled in two-dimensions and drawn to scale superimposes on the acceptable-area for the pyrimidine/parallel (shown in Figure 17(a)) and T and G/parallel (shown in Figure 17(b)) motifs.
  • F3 is in the syn-configuration with respect to the ribose.
  • the end of the F3 glycosyl bond is denoted by a square with an x to signify that F3 may be synthesized to have its backbone either parallel or antiparallel to the center strand (with the thymine base) .
  • the parallel orientation applies.
  • the F3 residue was positioned by placing the third-strand H-bonds at distances indicated in Table 7 for the types of H-bond.
  • Figure 18 depicts molecular modelling, drawn to scale, of. the 2,6 diaminopurine (D) residue to form a D:AT triplet that is more stable than the A:AT canonical base triplet in the purine/parallel and T and G/parallel motifs. Comparing to Figure 3, it is apparent that replacement of the hydrogen at C 2 of the third-strand adenine with an amino group to make 2, 6-diaminopurine allows an additional NH N strong H-bond with the N of the adenine of the direct AT base-pair. The resulting triplet, D:AT has substantially the same geometry as A:AT and will be energetically more favorable than A:AT.
  • the designed residue D can serve as a more stable replacement for A in D:AT in the purine/parallel motif where the r D , ⁇ , and ⁇ parameters are all in the preferred range. It can also serve in the purine/antiparallel and T and G/parallei motifs, where r D and ⁇ are in the preferred range and ⁇ is in the acceptable range, and perhaps also in the T and G/antiparallel motif, where r D and ⁇ are in the preferred range, and ⁇ lies just outside the edge of the acceptable range but within experimental error of the acceptable range.
  • Figure 19 depicts parallel-backbone-orientation molecular modelling, drawn to scale, of the base hypoxanthine in the nucleoside inosine (I) that is known to bind to both AT and GC direct base pairs.
  • the I: T and I:GC triplets modelled in two dimensions are shown in (a) and (b) , respectively.
  • the third-strand binding code states that the I nucleoside recognizes both AT and GC.
  • Figure 20 depicts the designed two-ring heterocycle residue, designated F4, binding to the inverted CG base pair in the purine/parallel, purine/antiparallel, T and G/parallel and T and G/antiparallel motifs, depending on whether the residue is oriented syn or anti about the sugar.
  • r D and ⁇ are the same and are in the preferred range, while ⁇ is in the acceptable range.
  • the end of the. F4 glycosyl bond is denoted by a square with an x to signify that F4 may have its backbone either parallel or antiparallel to the center strand (with the cytosine base) .
  • ' both orientations apply.
  • the F4 residue was positioned by placing the third-strand H-bonds at distances indicated in Table 7 for the types of H-bonds involved.
  • Figure 21 depicts the designed three ring heterocycle residue of Figure 14B of U.S. Patent No. 5,405,938 (designated SW) that, utilizing sugar-phosphate backbones or backbone analogs, will not bind to the inverted TA base pair in the pyrimidine/parallel motif, contrary to what was asserted in that patent.
  • SW TA triplet modelled in two dimensions and drawn to scale does not superimpose on the acceptable area for the pyrimidine/parallel motif.
  • a major object of the present invention is to circumvent the purine-rich target restriction, thus enabling third strands to bind to duplexes with core or center strands comprising mixed purine/pyrimidine sequences, with the consequence that either strand of the duplex could serve as the center strand and so define the target sequence.
  • the center or core strand is taken to be the duplex strand which is purine rich in the target sequence, i.e., has greater than 50% purines in the target sequence. If both duplex strands contain exactly 50% purines in the target sequence, then either strand may be designated the center or core strand.
  • the invention particularly provides processes for designing residues that when incorporated into third-strand oligonucleotides or their backbone analogs will bind specifically to "inverted" base pairs; i.e., base pairs with pyrimidines in the designated center strand (which is not allowed under the binding code governing standard bases and inosine, Table 1) .
  • Inverted base pairs therefore, are those that interrupt the runs of purine residues in the center target strand.
  • Another object of the invention is to design novel residues that bind more stably to "direct" base pairs (i.e., base pairs to which the standard bases and inosine bind as allowed by the binding code in Table 1) .
  • the designed residues sometimes bind to both bases in the duplex, not just the base in the center strand.. Indeed, according to the present invention, residues designed to bind to inverted base pairs must bind to both bases of the target base pairs.
  • a useful notation to describe third strands and direct and inverted base pairs is to let Z signify a standard or canonical third-strand base; R the standard or canonical purine bases A and G, and Y the standard or canonical pyrimidine bases T and C in the base pairs of a target duplex. Then, Z:RY denotes a base triplet with base Z bound to a "direct" base pair, that is, a base pair with a purine in the designated center strand. Furthermore, letting W signify a residue designed in accordance with the present invention, then W:YR denotes the base triplet with residue W bound to an "inverted" base pair, and W:RY denotes the base triplet with residue W bound to a "direct” base pair. Since the standard nucleic acid bases and inosine do not bind strongly to inverted base pairs, Z:YR denote "mismatches".
  • An inverted base pair is formally defined and obtained by rotating by 180° the H-bonded complementary base pair RY about the axis that is the perpendicular bisector of the line between the Ci' ends of the two glycosyl bonds.
  • the inverted AT and GC base pairs resulting from the rotation process which are therefore TA and CG, are illustrated in Figure 8.
  • the base pairs retain their geometry as required, but the pyrimidine base now resides on the designated center or core strand, which provides a base-pair target for designed residues W for triplexes with mixed purine-pyrimdine sequences in the center strand.
  • these inverted base-pair targets are denoted as YR, and resulting triplets as W:YR.
  • the present invention utilizes the fact that both the canonical AT/U and GC direct base-pairs and their inverted counterparts in standard A- or B-type duplexes have known, substantially-identical stereochemistry regarding glycosyl-bond location and orientation, and are substantially coplanar.
  • the designed third-strand residues form base triplets that are also substantially coplanar.
  • Substantially coplanar includes the propeller twist of the base pairs that does not affect the two-dimensional modelling described below.
  • third strands can be designed to bind stably and specifically to target duplexes with strands of mixed purine and pyrimidine sequences, and therefore to substantially any sequence.
  • the two-dimensional rules and guidelines for such design purposes are easy to visualize and implement, compared to the complex design required in three dimensions. Furthemore, the rules and guidelines define families of compositions of matter (residue families) , and within the families, the rules and guidelines define particular compositions of matter (residues) either novel or previously reported in the literature.
  • rules are design principles that must be followed; and if violated, the residue under consideration when incorporated in the third strand may not bind stably or specifically to its target inverted or direct base pair.
  • Guidelines are design concepts that, if followed, allow for the design and inclusion of residues that stably and/or specifically bind to its target direct or inverted base pair. However, it may be possible to design third-strand residues if one or more of the guidelines are not conformed to, although the resulting triplex is expected to be less stable.
  • the complete design process proceeds as follows: (1) Choose the third-strand binding motif (Table 2) for which the residue is to be designed. (2) Determine the planar ' molecular framework to which hydrogen bonding substituents or functional groups are to be added in conformity with the two-dimensional rules and guidelines for that motif. (3) Complete the design either by database searching for molecules with the desired molecular framework and appropriate functional groups or by constructing a novel residue with the desired molecular framework and appropriate functional groups. (4) Confirm the design using two-dimensional and three-dimensional space-filling models and energy minimization techniques. In this design process, three-dimensional space-filling modelling is used only to confirm a design, a much simpler use of 3D modelling than de novo design. (5) Synthesize the residue and incorporate into a third-strand oligonucleotide to quantify its stability and specificity in the appropriate triplex test system for that motif.
  • RY or YR may be direct or mediated by no more than one H 2 0 molecule.
  • H-bonds between W and RY Rule or YR may include non-standard and non-linear H-bonds, but at least one and preferably two, standard substantially-linear H-bonds must be present.
  • the core target duplex exists in the A-type duplex form
  • some evidence has recently emerged that the core duplex in at least some triplex motifs may be of the B-type or somewhere between the two.
  • helix form only affects the way the backbone strands wind about each other and base pairs tilt and overlap; but it has no apparent impact on the H-bonding schemes and stereochemisty of the target base pairs themselves, or on the H-bonding schemes of the base triplets of triplexes (see 4. below).
  • the third strand regardless of its orientation (parallel or antiparallel) with respect to the purine-rich strand of the target duplex, lies in the major groove of the target duplex. Moreover, the ribose-phosphate backbone of the third strand has a sufficient number of rotatable bonds in each residue to adjust to the H-bonding requirements of each of the observed triplex motifs.
  • the residues Z or W can H-bond, depending on the triplex motif (and the structure of W) , either only to N 7 and the C ⁇ substituent of the purine member of a direct target base pair; or to either or both these sites and to the C ⁇ substituent of the pyrimidine member of such a pair. Further, Z or W can H-bond to the C 4 substituent of the pyrimidine member of an inverted target base pair and at least to the Cg substituent of the purine member of the target pair.
  • a glycosyl bond is defined in its broadest aspect to be the bond between.a base or residue and a backbone.
  • the glycosyl bond is the bond between the Cl' sugar atom and either the N9 purine or NI pyrimidine atoms.
  • a residue with a native backbone it is the bond linking the Cl' sugar atom to the residue.
  • a residue with other than a native backbone it is the bond between the residue and the backbone.
  • both canonical base triplets should have substantially the same D-circle center and radius, as is the case in the pyrimidine/parallel and in the purine/parallel motifs (see Figures 2 and 3) .
  • the two canonical base triplets lie on D circles of somewhat different centers and radii.
  • the three points located at the Ci' ends of the three glycosyl bonds lie on the circumference of a unique circle, that is, the D circle of the invention.
  • the center of the D circle is located at the intersection of the perpendicular bisectors of any two of the three lines connecting the three Cl' points.
  • the ⁇ ranges for the canonical base triplets are determined from the deviations tolerated in canonical duplexes and triplexes, including modelling experimental error.
  • the acceptable ranges were determined by evaluating ranges allowable in the five known motifs, and performing two-dimensional modelling with different acceptable H-bond distances and angles, so as to avoid steric hindrance and strain in the backbone.
  • the preferred ranges were picked to be close to the canonical base triplets.
  • Table 5 below presents the clockwise angular displacement values ⁇ (on the D circle) of the Cl' atom attached to the Z base from the purine (R) Cl' sugar atom of a direct target base pair RY in a canonical triplet Z:RY in each motif (see Figure 7) .
  • the ⁇ ranges for the canonical base triplets were determined from the deviations tolerated in canonical duplexes and triplexes, including modelling experimental error.
  • the acceptable ranges for W residues were determined by evaluating ranges allowable in the five known motifs, and performing two-dimensional modelling with different acceptable H-bond distances and angles so as to avoid steric hindrance and severe strain in the backbone. The preferred ranges were picked to be close to the canonical base triplets.
  • the glycosyl bond for each residue W should either point substantially outward from or substantially tangential to the D-circle. If substantially tangential, it should point toward the empty space in the D-circle to eliminate the possibility of steric interference between backbone atoms air, base-triplet atoms of neighboring triplets.
  • the gl! conjunction ..osyl bond direction is that pointing from the Ng atom of the purine base to the Ci' atom of the sugar, or from the Ni atom of the pyrimidine base to .the Ci' atom of the sugar.
  • the Ng and Ni atoms are denoted only as N (for nitrogen) to preserve the geometrically accurate two-dimensional representation.
  • Figure 1 illustrates the usual numbering scheme for purines and pyrimdines.
  • ⁇ is the angle (measured clockwise) formed between the D-circle radius and the glycosyl bond vector of the Z or W third-strand residue (see Figure 7) .
  • the ⁇ angles may be observed in Figures 2-6, and the values and ranges are presented in Table 6 below.
  • the preferred range of values are substantially the same as those for the canonical base triplets, Z:RY, but acceptable values include all outward pointing glycosyl bonds (low values of ⁇ ) to values tangential to the circle (values of ⁇ near +90° or -90°) provided there is no steric hindrance.
  • T and 0,1 T AT G:GC -30 to +120°
  • the ⁇ ranges for the canonical base triplets take account of the deviations tolerable in canonical duplexes and triplexes and also the experimental error of modelling.
  • the preferred ranges are those nearer the values for the canonical third-strand bases.
  • the acceptable ranges include the preferred ranges plus essentially any angle that will not cause steric hindrance between the backbone and the third-strand residues.
  • Stable triplex formation will occur when: a) the third-strand residue W of a base triplet is H-bonded optimally to the direct or inverted base pair; b) the glycosyl bond has a radius in the preferred or acceptable range (Table 4) and has a preferred or acceptable position (Table 5) ; c) its glycosyl bond direction is in the preferred or acceptable range (Table 6) ; and d) the other rules and guidelines are adhered to. If the direction of the glycosyl bond of the designed W residue is outside the acceptable range for that motif, triplex formation is unlikely.
  • the molecular framework for W should be substantially planar and sterically compatible with the van der Waals thickness of one base pair in a nucleic acid helix; i.e., about 3.4 A.
  • the dimensions of the framework and the location of H-bond donor and acceptor substituents must allow H-bond formation with the direct or inverted base-pair targets.
  • the D-circle radius on which the glycosyl bond of W lies should fall within the preferred or acceptable ranges defined in Table 4; and that glycosyl bond should fall within the range of positions and orientations defined in Tables 5 and 6 for the various motifs.
  • heterocyclic and nonheterocyclic molecular structures all within the scope of the invention, can be devised, modelled, and synthesized that fulfill the framework, glycosyl-bond location, and H-bonding requirements
  • carbon-nitrogen heterocycle frameworks are, a priori, the simplest choices.
  • Novel carbon-nitrogen heterocycle frameworks with two, three and four rings that provide aromaticity and -structural planarity, rigidity, and molecular dimensions compatible with preferred D-circle sizes, are preferred compositions of matter in the invention.
  • These two, three and four ring frameworks are preferred also because they provide a number of ring nitrogens to serve as H-bond donors, acceptors, a number of positions for N-Ci' glycosyl bonds, and reactive ring atoms to which hydrogen-bonding donor and acceptor functional groups (or functional groups capable of other weak interactions) may be attached.
  • FIG. 14 Two examples of such three ring heterocycles, one of a four-ring heterocycle, and one of a two-ring heterocycle are shown in Figure 14.
  • the ring positions of the functional groups may be other than those shown in the three specific examples of the figure.
  • These novel frameworks are designated W F" to differentiate them from the wide range of frameworks and residues W that are within the scope of the invention.
  • compositions of matter based on the framework will be denoted by Fl, F2, F3, F4, etc. below.
  • the three and four ring heterocycle F frameworks and F-residues are preferred embodiments of the invention. These include the carbon-nitrogen ring heterocycles in the Examples and may also include rings containing other atoms such as O, S, etc.
  • the chemical groups (here usually called functional groups, or substituents) adorning the framework of W should lie (or be able to lie) preferably in the same plane as the framework or substantially no more distant from the plane than those of the canonical bases.
  • the -standard H-bonds listed with their preferred bond distances in Table 7 below are the preferred functional groups in the invention, although other sources of H-bond acceptors such as fluorine atoms are known that can be substituted on the framework (L. Pauling, Nature of the Chemical Bond, Cornell University Press, 3rd ed. , p. 454 (I960)) .
  • certain complementarily-sized hydrophobic substituents on the framework can also provide binding energy alternatives to that provided by H-bonding.
  • H-bonding substituents When bound to ring atoms of heterocycles, some H-bonding substituents conform to the planar restriction (in some cases, when rotated about their single bonds into positions compatible to H-bonding within the plane) . Also, in some cases, an H-bond donor can rotate its substituent to direct its donor H-atom to an acceptor in the nearest-neighbor base pair above or below the opposing target base pair.
  • Table 7 below presents standard H-bonding functional groups and the H-bonds they form.
  • the ⁇ values indicate the typical range, and therefore the preferred range, of distances from the center of the donor atom of the H atom to the center of the acceptor atom.
  • the H-bonding target for W is either the R base or both bases.
  • W must form at least two H-bonds with a target RY or YR base pair if other substantial weak interactions between the base pair and the W residue are not present.
  • the W residue must bind to both bases in the pair since only one H-bonding site is available on the Y base (see Figure 8) . Otherwise, the glycosyl bond D-circle, position and direction guidelines will not be met.
  • Hydrogen bonds are mainly electrostatic in nature. Compared with covalent bonds of well-defined length, strength and orientation, hydrogen bonds are "soft" and only weakly directional (Saenger, W. , in Principles of Nucleic Acid Structure, Springer-Verlag New York Inc. (1984)). Three novel types of H-bonds, along with conventional ones, have been recognized in nucleic acid and nucleoprotein interactions: the carbon donor CH O and
  • both conventional and non-standard H-bonds are considered in deducing possible H-bonding schemes with at least two H-bonds between a third strand base or residue and a direct or inverted base pair.
  • fluorine substituents on frameworks are considered as acceptors for donated H atoms (L. Pauling, Nature of the Chemical Bond, Cornell University press, 3rd ed. , p. 454 (I960)).
  • H-bonds between W and RY or YR may be mediated by no more than one H 2 0 molecule; and (2) H-bonds between W and RY or YR may include non-standard and non-linear H-bonds, but at least one standard, linear H-bond must be present. Less than 25° bending from linearity is considered linear in the practice of the invention, that is, will form a strong H-bond.
  • Non-standard H-bonds in place of one of the two desired H-bonds such as those involving carbon-hydrogens, e.g., CH--N, CH---0, CH 3 ---N, CH 3 ---0 are not to be discounted.
  • the 1-acc. position and the 2-don. position lie in the same relative positions, but they are not " identically located with respect to the base-pair glycosyl bonds.
  • positions 1 have a donor and positions 2 have an acceptor, so that they lie in the same relative locations with respect to the base-pair glycosyl bonds, but not at the identical locations with respect to the base pair glycosyl bonds.
  • a designed residue that binds only to its target inverted base pair may be said to give "full” specificity, whereas a designed residue that binds also to a direct base pair may said to give "partial” specificity.
  • a designed residue with full specificity can bind only to one of the four target base pairs, whereas with partial specificity it can bind to two of the four target base pairs.
  • what counts is the specificity of the entire third-strand, not just the specificity of any particular third-strand residue.
  • compositions of the present invention include both native and artificial backbones.
  • Backbones that have flexibility similar to native sugar-phosphate backbones and that have been shown to allow the formation of stable duplexes or triplexes are useful in the invention.
  • the stereochemistry of base triplets and acceptable H-bonding schemes of the five experimentally-verified motifs determine acceptable backbone locations on the D-circle. These motif experiments employed native sugar-phosphate backbones.
  • Backbones may not be too stiff or too flexible.
  • a backbone that is too stiff may not be able to conform to acceptable ranges for r D , ⁇ and ⁇ .
  • a backbone that is too flexible may undergo a large negative entropy change, leading to an unfavorable positive free-energy change, upon triplex formation.
  • a simple, acceptable measure of flexibility is the number of rotatable backbone bonds between third-strand bases on adjacent triplets.
  • the native sugar-phosphate backbones have six rotatable bonds (i.e., the 4'C-3'C; 3'C- O; O-P; P-O; O-5'C and 5'C-4'C bonds).
  • phosphorothioate and methylphosphonate also have six rotatable bonds, and so are preferred backbones in the invention along with the native sugar-phosphate backbones.
  • Other backbones with one or two more or one or two less rotatable bonds than the native sugar-phosphate backbone are still acceptable in the invention, provided that they have been shown to allow formation of stable triplexes. In fact, any backbone shown to form stable triplexes is acceptable.
  • a defining characteristic of each third strand binding motif is the polarity of the third strand relative to the center or core strand of the target duplex.
  • the standard definition of parallel and antiparallel refers to whether the two strands of a duplex are oriented in the same (parallel) direction, both 5' to 3' , or in opposite (antiparallel) directions, 5' to 3' and 3' to 5'.
  • the two strands are oriented antiparallel, and the bases attached to each strand are in the anti configuration about their glycosyl bonds.
  • third strand binding with canonical bases and W residues would be limited by ionization of ring phenolic OH substituents adjacent to unsubstituted ring nitrogens, which generally have a pK a 9-9.5.
  • a pH of approximately 8-8.5 generally represents an upper pH limit to third strand binding.
  • ionization of unsubstituted ring nitrogens serving as hydrogen acceptors for H-bonding have pKa values near 4.5 or below, so pH values below about 5 should generally be avoided.
  • Ionic Strength In general, third strand binding is favored by increasing ionic strength, with 0.01 M monovalent cation at room temperature serving as a lower limit. Addition of Mg 2+ to about 5-20 mM is generally very stabilizing, as is increasing monovalent cation alone up to about 1 M. The preferred monovalent cation is sodium, which does not encourage tetraplex formation of G rich strands, as does potassium ion.
  • Triplexes with adequate stability for particular purposes can be as short as about 7-10 base triplets in length, although for purposes of gene therapy by induced mutagenesis or by suppression of gene expression or of replication, lengths of 15-25 base triplets are required to limit third strand binding to unique targets in the human and other large genomes .
  • the motifs have been described by reference to the base composition of the third -strand (i.e., purine, pyrimidine or T and G) , it will be understood that the third strand need not contain only those bases.
  • the frequency of determinative- ' bases and/or base analogs plus synthetic residues is no less than five out of seven.
  • the present invention is directed to circumventing the requirement that third strand binding targets comprise strands that are very purine-rich pyrimidine-rich duplex sequences. This achievement in no way reduces the utility of third strand binding as described in U.S. Patent No. 5,422,251 to Fresco. .Rather, it enhances the utility by making virtually all Watson-Crick or complementary duplex sequences accessible to third strand binding. Presently, less them one percent of the genomic sequences -of essentially all living organisms, including viruses, bacteria, invertebrates and vertebrate organisms, including mammals and particularly humans, are accessible to third strand binding.
  • this invention provides the basis for a quantum leap in the utility of such binding for control of replication and gene expression in all living organisms; for inducing mutations; for the repair of mutations in vitro and in vivo; including applications resulting in gene therapy, the inhibition of infectious bacteria and viruses, the creation of phenotypic mutations; for diagnostic purposes; for isolating specific double stranded nucleic acid fragments, including chromosomes, YACS, other types of DNA particles, restriction fragments; and for the development of new types of specific third strand DNA probes for diagnostic and research purposes.
  • an example is provided of the design of a third strand to the purine-rich target site present at the site of the A—>T substitution mutation in ⁇ globin that causes sickle cell anemia.
  • This example has utility as a means of specifically correcting the mutation by utilizing a third strand with a psoralen moiety covalently bound to it at the position corresponding to the A—>T mutation.
  • Psoralen damage to a T base in chromosomal DNA in eukaryotes is often misrepaired to an A base, just the change required here to revert the sickle-cell ⁇ globin gene to the normal ⁇ globin gene.
  • This therapeutic application is the subject of copending U.S. patent application to Glazer, filed concurrently herewith.
  • the codons "gag” and “gug” code for glutamic acid and valine, respectively.
  • the underlined DNA region is the target for third-strand binding.
  • the "/" indicates a strand switch; bold letters indicate bases opposite mismatch sites (pyrimidines in the purine-rich target strand) ; and "pso" indicates the attached psoralen opposite the T mutation.
  • the left-hand sequence (before the first strand switch) is in the pyrimidine/parallel motif; the center sequence is in the G and T/antiparallel motif; and the right-hand sequence is again in the pyrimidine/parallel motif.
  • This example illustrates the utility of only a single W designed residue targeted to only one of the two inverted base pairs (CG) .
  • compositions of matter designed by the processes of the invention are provided. It is understood that the examples are not intended to limit the invention and that other embodiments of the invention will be apparent from the information provided to those of ordinary skill in the art.
  • the glycosyl bond is attached to Ng, and the base is in the anti-configuration with respect to the sugar.
  • the end of the Fl glycosyl bond is denoted by a square with an x to signify that Fl may be synthesized to have its backbone parallel or antiparallel to the center strand, and here the parallel orientation applies.
  • ⁇ 82°
  • the end of the F3 glycosyl bond denoted by the square with an x
  • the glycosyl bond is attached to the N atom of the ring at the extreme left. All four rings, with one ring in the extreme left position are necessary to bring the position of the glycosyl bond into conformity with the pyrimidine/parallel motif.
  • the end of the F3 glycosyl bond is denoted by a square with an x to signify that F3 may be synthesized to have its backbone parallel or antiparallel to the center strand, and here the parallel orientation applies, with F3 in the syn configuration with respect to the sugar.
  • the F3 residue was positioned by placing the third-strand H-bonds at the median distances indicated in Table 7 for that type of H-bond.
  • the F3 residue can be prepared according to Scheme 2, below.
  • Removal of the MOM protecting groups by HCl in ethanol, followed by chlorination with PCI 5 and reduction yields VTII.
  • After saponification of VIII, the free acid will be subjected to Arndt-Estert reaction to extend one carbon unit to give IX in which the methylene group is activated.
  • the designed residue D can serve as a more stable replacement for A in D:AT in the purine/parallel motif where the r D , ⁇ , and ⁇ parameters are all in the preferred range, and in the purine/antiparallel and T and G/ parallel motifs where r D and ⁇ are in the preferred range and ⁇ is in the acceptable range, and perhaps in the T and G/parallel motif where r D and ⁇ are in the preferred range, and ⁇ lies just outside the edge of the acceptable range but within experimental error of the acceptable range (see Tables 4, 5 and 6) .
  • a phosphoramidite precursor of D, S6-dinitrophenol-2' deoxyguanosine ⁇ -cyanoethylphosphoramidite with DMT and dimethylformamidine protecting groups is commercially available that, after incorporation by solid phase synthesis into a potential third strand of appropriate sequence, can be quantitatively deblocked by standard methods to give the desired third strand containing the D residue(s) .
  • the third-strand binding code (Table 1) states that the I base recognizes both AT and GC base pairs.
  • the experiments of Letai, et al . show that I forms triplexes with homopolymer GC duplexes, homopolymer AT duplexes, and mixed base-pair duplexes with alternating AG in the center strand. Because of the base-sequence symmetry with regard to backbone orientation of. homo-duplexes and alternating duplexes, it is not known whether the third strands bind in the parallel, antiparallel, or both orientations.
  • the modelling indicates that both stable I:AT and I:GC triplets can form in the parallel orientation.
  • I: T and I:GC triplets were also modelled in the antiparallel orientation (illustration not presented) .
  • the similarity of these values between I:AT and I:GC indicate that third-strands of I-homopolymer will form; however, the large 0 values for both I:AT and I:GC preclude the use of I in place of A and G residues of any third strands to bind in the antiparallel orientation, as the ⁇ values fall outside the acceptable ranges for any of those motifs.
  • the F4:CG triplet is modelled in two dimensions ( Figure 20) .
  • r D 8.0 A
  • 0 101°
  • ⁇ -32°
  • r D and 0 are in the preferred range
  • ⁇ is in the acceptable range.
  • the F4 residue may be synthesized by the procedure outlined in Scheme 1, below. Treatment "of cytosine (I) or cytidine (II) with malonyl chloride (III) in pyridine gives the pyrimido[1,2-a]pyrimidine (IV), which may be treated with ammonia to yield the product (V) . As with the other examples of F residues, this two ring system is readily convertable to phosphoramidites for solid phase synthesis of a desired third strand.
  • the third strand residue shown in Figure 14B of U.S. Patent No. 5,405,938 was modelled as a third strand residue for the pyrimidine/parallel motif.
  • the result is shown in Figure 21.
  • the SW residue was positioned by placing the third strand H-bonds at distances indicated in Table 7 for the types of H-bond.
  • This residue of U.S. Patent No. 5,405,938 should not form a stable triplet in the pyrimidine parallel motif with any native sugar- phosphate or analog backbones.

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Abstract

La présente invention concerne des règles et des principes permettant de concevoir des bases hétérocycliques qui, lorsqu'elles sont incorporées dans des oligonucléotides, permettent à ces oligonucléotides de servir d'acides nucléiques de troisième brin capable d'une liaison spécifique avec les acides nucléiques à double brin de n'importe quelle séquence paire de base, sans qu'il y ait besoin de brin riche en purine. Les bases servant d'exemple de la figure ont été conçues en mettant en ÷uvre le procédé de l'invention.
PCT/US1996/009428 1995-06-07 1996-06-06 Restes permettant de lier des troisiemes brins a des complexes doubles nucleotidiques complementaires de n'importe quelle sequence paire de base WO1996041009A1 (fr)

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AU62601/96A AU6260196A (en) 1995-06-07 1996-06-06 Residues for binding third strands to complementary nucleic acid duplexes of any base pair sequence
EP96921358A EP0871772A4 (fr) 1995-06-07 1996-06-06 Restes permettant de lier des troisiemes brins a des complexes doubles nucleotidiques complementaires de n'importe quelle sequence paire de base

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US08/473,888 US6426407B1 (en) 1995-06-07 1995-06-07 Residues for binding third strands to complementary nucleic acid duplexes of any base pair sequence
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Cited By (2)

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WO1999024622A1 (fr) * 1997-11-10 1999-05-20 Princeton University Hybridation in-situ triplex
WO2012011114A3 (fr) * 2010-07-22 2012-07-26 Genearrest Ltd Composés de liaison à l'adn/arn double brin spécifiques de certaines séquences et leurs utilisations

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US7309569B2 (en) * 1999-12-21 2007-12-18 Ingeneus, Inc. Parallel or antiparallel, homologous or complementary binding of nucleic acids or analogues thereof to form duplex, triplex or quadruplex complexes
US7220541B2 (en) * 2000-01-24 2007-05-22 Ingeneus, Inc. Homogeneous assay of biopolymer binding by means of multiple measurements under varied conditions
BRPI0409777A (pt) * 2003-04-30 2006-05-30 Pharmacia Corp compostos que apresentam uma parte bicìclica fundida para ligação com o sulco menor de dnadf

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JP3495752B2 (ja) * 1992-12-11 2004-02-09 キヤノン株式会社 生標的個体の検出方法およびプローブ

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WO1992009705A1 (fr) * 1990-11-23 1992-06-11 Gilead Sciences, Inc. Oligomeres formant un triplex et contenant des bases modifiees
US5352580A (en) * 1993-05-26 1994-10-04 Becton, Dickinson And Company Selective detection of mycobacteria by nucleicacid probes derived from Mycobacterium kansasii

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ANN. REV. BIOPHYS. BIOMOL. STRUCT., 1995, Vol. 24, PLUM et al., "Nucleic Acid Hybridization: Triplex Stability and Energetics", pages 319-350. *
BIOCHEM. SOC. TRANS., October 1989, Vol. 17, No. 5, VOSS et al., "Use of 2,6-Diaminopurine in Oligonucleotide Gene Probes", page 913. *
BIOPOLYMERS, 1993, Vol. 33, MOHAN et al., "Molecular Recognition of Watson Crick Base-Pair Reversals in Triple Helix Formation: Use of Nonnatural Oligonucleotide Bases", pages 1317-1325. *
BIOPOLYMERS, 1995, Vol. 35, CHENG et al., "Solvent Effects on Model d(CG G)7 and d(TA T)7 DNA Triple Helices", pages 457-473. *
J. AM. CHEM. SOC., 1994, Vol. 116, XIANG et al., "A New Pyrimidine Nucleoside (m50xC) for the pH-Independent Recognition of G-C Base Pairs by Oligonucleotide-Directed Triplex Formation", pages 11155-11156. *
MOL. ENGINEER., 1995, Vol. 5, SUN et al., "Rational Design of Switched Triple Helix Forming Oligonucleotides: Extension of Sequences for Triple Helix Formation", pages 157-178. *
NUCLEIC ACIDS RES., 1988, Vol. 16, No. 1, CHOLLET et al., "DNA Containing the Base Analogue 2-Aminoadenine: Preparation, Use as Hybridization Probes and Cleavage by Restriction Enzymes", pages 305-317. *
NUCLEIC ACIDS RES., 1988, Vol. 16, No. 11, CHEONG et al., "Thermodynamic Studies of Base Pairing Involving 2,6-Diaminopurine", pages 5115-5122. *
NUCLEOSIDES NUCLEOTIDES, 1994, Vol. 13, No. 1-3, CANO et al., "Synthesis of Oligodeoxyribonucleotides Containing 2,6-Diaminopurine", pages 501-509. *
See also references of EP0871772A4 *
SEKHARUDU et al., "DNA Triple Helices with Reverse-Hoogsteen and Purine-Purine Hydrogen Bonding: A Molecular Dynamics Study", In: STRUCTURAL BIOLOGY: THE STATE OF ART, PROCEEDINGS OF THE EIGHT CONVERSATION, Edited by R. SARMA et al., NEW YORK: ADENINE PRESS, 1994, pages 113-125. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999024622A1 (fr) * 1997-11-10 1999-05-20 Princeton University Hybridation in-situ triplex
WO2012011114A3 (fr) * 2010-07-22 2012-07-26 Genearrest Ltd Composés de liaison à l'adn/arn double brin spécifiques de certaines séquences et leurs utilisations

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